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Effect Of Metabolites Of Heme Oxygenase-1 On Bovine Viral Diarrhea Virus Infection In MDBK Cells

Posted on:2017-08-13Degree:MasterType:Thesis
Country:ChinaCandidate:F X PuFull Text:PDF
GTID:2370330485480776Subject:Prevention of Veterinary Medicine
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Bovine viral diarrhoea(BVD)is one of the most economically important viral diseases affecting the cattle industry worldwide.The etiologic agent of BVD is BVD virus(BVDV).However,current antiviral strategy could not effectively prevent and control this disease.Therefore,it is imperative to study BVDV biology for the development of antiviral strategies to combat BVDV infection.Heme oxygenase-1(HO-1),an important anti-oxidative stress protein,have numerous biological functions including anti-oxidant,anti-inflammatory,anti-apoptotic and anti-proliferative properties.Recent studies have also demonstrated that HO-1 has significant antiviral properties.Our previous work showed that induction of HO-1 expression inhibits significantly BVDV replication.However,the molecular mechanisms of HO-1 in the inhibition of BVDV replication is unknown.HO-1 catabolizes free heme into biliverdin,carbon monoxide(CO)and iron.In this study,we researched the effect of CO,bliverdin and iron on BVDV replication.The proposed studies are expected to provide additional information for better understanting the molecular mechanisms of HO-1 in inhibiting BVDV replication.The main works and results were as follows: 1.CO suppresses markedly BVDV replication in MDBK cellsTo confirm the effect of CO on BVDV replication,MDBK cells infected with BVDV were treated with different concentrations of CORM-2 from 1hpi.Then 24 hpi cells and supernatant were harvested to determine intracellular BVDV mRNA and protein expression,copies of virus genome and virus titer in the supernatants by qPCR,Western blot and TCID50.The results showed that CORM-2 treatment attenuates BVDV replication in a dose-dependent manner in MDBK cells.Further,CORM-2 treatment inhibits BVDV replication in a time-dependent manner.At the same time,CORM-2 treatment also inhibits different MOI of BVDV replication in MDBK cells.To determine whether CORM-2 affects BVDV virulence through reducing virus infectivity after direct contact with the virus,BVDV and different concentrations of CORM-2 were co-incubated for 1h at 37?.Virus titers were determined in MDBK cells.The results showed that CORM-2 is virucidal at the concentration of 150?M.These results indicate that CORM-2 treatment attenuates BVDV replication via both direct virucidal properties and intracellular regulation.2.Biliverdin inhibits significantly BVDV replication in MDBK cellsTo determine the effect of biliverdin on BVDV replication,MDBK cells were incubated with the indicated concentrations of biliverdin.The results showed that biliverdin treatment markedly reduced intracellular BVDV RNA,and intracellular BVDV protein,the levels of BVDV RNA in the supernatants and virus titers in a dose-dependent manner.Then,we investigated the BVDV growth dynamics in MDBK cells treated with biliverdin.influence of different times between transfection and infection on the effect of inhibition.The results showed that viral growth was inhibited much more significantly in MDBK cells infected with BVDV at 12 hpi than other time points.To determine whether biliverdin affects BVDV virulence through reducing virus infectivity after direct contact with the virus,BVDV and different concentrations of biliverdin were co-incubated for 1h at 37?.Virus titers were determined in MDBK cells.The results showed that there was no significant difference between virus treated with biliverdin and control,indicating biliverdin is not virucidal at the concentrations used in this study.To determine the stage(s)of the BVDV life cycle that is targeted by biliverdin,we investigated the effect of biliverdin on BVDV adsorption and penetration.The results indicate that biliverdin had no effect on BVDV adsorption,but it significantly affect BVDV penetrating into MDBK cells.3.Iron does not affect BVDV replication in MDBKsTo confirm the effect of iron on BVDV replication,MDBK cells infected with BVDV were treated with different concentrations of FeCl3 from 1hpi.Then 36 hpi cells and supernatant were harvested to determine intracellular BVDV mRNA and protein expression,and virus titer in the supernatants by qPCR,Western blot and TCID50.The results showed that there was no significant effect of iron on BVDV replication in MDBK cells.In summary,these data suggest that HO-1-dependent inhibition of BVDV replication is partially dependent upon both CO and biliverdin but independent on iron.The results generated from the studies provide additional information for better understanting the molecular mechanisms of HO-1 in inhibiting BVDV replication and will pave the way for developing HO-1 and its metabolites based novel approach for the prevention and control of BVD.
Keywords/Search Tags:Bovine viral diarrhea virus(BVDV), Heme Oxygenase-1(HO-1), CO, Biliverdin, Iron
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